Von Willebrand factor (VWF) is a multimeric blood glycoprotein that is required for hemostasis. At sites of vascular injury, VWF binds connective tissue and then mediates platelet adhesion through binding to platelet membrane glycoprotein lb. The adhesive function of VWF depends on the largest multimers, and inability to assemble multimers causes bleeding associated with von Willebrand disease. Conversely, VWF multimers are cleaved at sites of thrombosis by ADAMTS13, a metalloprotease, and ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. Therefore, the control of VWF multimer size has substantial medical importance, and understanding the function of VWF depends on understanding the assembly and structure of VWF multimers. In the endoplasmic reticulum, proVWF subunits dimerize through their C-terminal CK domains, and Specific Aim 1 is to characterize the intersubunit disulfide structure of the dimeric VWF CK domain. ProVWF dimers are transported to the Golgi, where the propeptide promotes the formation of additional intersubunit disulfide bonds between N-terminal VWF D'D3 domains, thereby forming multimers, and Specific Aim 2 is to characterize the intrachain and interchain disulfide bonds of the multimerization site in the VWF D3 domain. The disulfide bonds of recombinant VWF CK dimers and recombinant VWF D'D3 dimers will be identified by partial reduction and alkylation, chemical and enzymatic fragmentation, protein sequencing, and mass spectrometry. Specific Aim 3 is to characterize the propeptide-dependent mechanism of VWF multimerization. The VWF propeptide appears to function as an endogenous disulfide isomerase that promotes intersubunit disulfide bond formation not in the ER but in the Golgi, where disulfides otherwise do not form, and this remarkable mechanism is conserved in a family of multimeric epithelial mucins. The mechanism of VWF multimer assembly will be investigated with novel assays for disulfide bond formation between recombinant VWF D'D3 domains. Disulfide-linked intracellular intermediates between VWF propeptide and D'D3 domains have been identified and will be characterized. The function of Cys residues that form interdomain and intersubunit disulfides will be determined by mutagenesis. The structural requirements for VWF multimerization will be characterized with chimeric constructs of human VWF and homologous mucins, and of interspecies VWF chimeric proteins.